To protect humans from pathogenic microorganisms, many antibiotics have been discovered, developed and used. However, the misuse of antibiotics has resulted in a rapid increase in antibiotic-resistant strains, and thus the number of usable antibiotics has been limited. For this reason, there has been a demand for novel substances which have activation mechanisms different from conventional antibiotics, exhibit activity against antibiotic-resistant microorganisms, do not cause problems on resistance and do not remain in vivo for a long period of time. Typical candidates capable of satisfying this demand include antimicrobial peptides.
Unlike conventional antibiotics, antimicrobial peptides have potent antimicrobial activities against a wide range of microorganisms, are physically and chemically stable in heat, acid or alkali and consist of a small number of amino acids (5-50 amino acids). Thus, these antimicrobial peptides have advantages in that they are easily degraded after antimicrobial action so that they do not remain in vivo, indicating that they do not cause toxicity in vivo. Thus, the antimicrobial peptides can be used as next-generation antibiotic substances and are highly applicable in industrial fields, including the pharmaceutical and food fields.
The present inventors previously developed antimicrobial peptides having potent antimicrobial activity against a wide range of microorganisms (Korean Patent Registration No. 0441402).
For industrial application of these antimicrobial peptides, methods capable of producing large amounts of the antimicrobial peptides in a cost-effective manner are by necessity required, but conventional methods for producing the antimicrobial peptides cannot provide large amounts of the antimicrobial peptides in a cost-effective manner. In other words, the use of a chemical synthesis method, which is a conventional method for peptide production has low economic efficiency, and when an antimicrobial peptide is produced from microorganisms using genetic engineering technology there are problems in that the antimicrobial peptide is expressed at a low level and shows antimicrobial activity against the host and in that the expressed antimicrobial peptide is easily degraded by proteinases in the host.
In addition, in order to highly express an antimicrobial peptide in microorganisms, a method of producing a desired peptide from host microorganisms using a fusion partner without killing the host cells was generally used in the prior art.
In the above method, in order to recover the antimicrobial peptide, it is required to lyse the host cell to obtain an insoluble fusion protein, digest the fusion protein and isolate and purify the antimicrobial peptide using a chromatography or ion-exchange column. However, the above method has a critical problem in that a large amount of the antimicrobial peptide is lost during the recovery process so that the yield thereof is significantly reduced, resulting in a significant increase in the price of the antimicrobial peptide.
To overcome this problem, an attempt was made to fuse a cell surface display protein with an antimicrobial peptide to display the antimicrobial peptide on the cell surface. As a result, the cell lysis process could be omitted by displaying the antimicrobial peptide on the cell surface, but there were still problems in that the cell surface display protein must be treated with a separate enzyme in order to isolate the antimicrobial peptide and in that a chromatography or ion-exchange column must be used to remove impurities.
In addition, there is a method in which an antimicrobial peptide displayed on the cell surface is used without any treatment in order to omit the process of isolating and purifying the antimicrobial peptide. However, this method has a serious problem in that the antimicrobial activity of the antimicrobial peptide attached to the cell surface is significantly reduced.